Aldoximes have the general chemical structure RHC═NOH where “R” is an aryl or alkyl group. Plants synthesize a variety of aldoximes as intermediates in biochemical pathways leading to compounds that are used as developmental signals and as defenses against pathogens (Hull et al., PNAS USA, 97:2379–84 (2000); Wittstock and Halkier, J Biol Chem, 275:14659–66 (2000)). A variety of microorganisms are known to degrade aldoximes (Kato et al., Appl. Environ. Microbiol., 66:2290–2296 (2000)). Bacteria typically degrade aldoximes by first converting the aldoxime to a nitrile by means of aldoxime dehydratase and subsequently converting the nitrile to a carboxylic acid by means of nitrilase or a combination of nitrile hydratase and amidase (FIG. 1). The ability of plants to synthesize various aldoximes and the possibility of converting an aldoxime into a nitrile, amide or carboxylic acid make aldoximes potentially useful as starting materials or as intermediates in biotransformations.
One factor that limits the use of aldoximes in biotransformations is the lack of identified aldoxime dehydratase genes. In deed, only one aldoxime dehydratase gene is known in the literature (Kato et al., Biochem 39:800–809 (2000)). Although several strains of Rhodococcus and other bacteria that degrade aldoximes and have aldoxime dehydratase activity are described in the existing literature (Kato et al., Appl. Environ. Micorbiol., 66:2290–2296 (2000)), with the exception of genes from Bacillus, the genes for aldoxime dehydratase in these bacteria are largely unknown. One report exists describing the cloning and expression of a gene encoding a phenylacetaldoxime dehydratase, from a Bacillus sp. strain OxB-1, and the recombinant production of arylalkyl and alkyl-nitriles from the corresponding aldoximes (Xie et al., Biosci. Biotechnol. Biochem., 65(12):2666–2672 (2001)).
The problem to be solved therefore is to identify new aldoxime dehydratase genes for use in the recombinant production of nitriles, amides, carboxylic acids, and downstream intermediates in the aldoxime-nitrile-carboxylic acid pathway.
Applicants have solved the stated problem by isolating the gene for aldoxime dehydratase (oxd) from a Rhodococcus erythropolis AN12 strain containing an open reading frame (ORF) that encodes aldoxime dehydratase and by expressing the Rhodococcus oxd gene in E. coli. 